ODN 2243 (ODN 2216 Control) - TLR9 ligand

Negative control of ODN 22016 - Class A CpG oligonucleotide - Human TLR9-preferred ligand

No TLR9 activation with ODN 2243 (ODN 2216 Control)

ODN 2243 (also known as ODN 2216 Control) is designed as a negative control for the TLR9 agonist ODN 2216. ODN 2216 is an immunostimulatory Class A CpG oligonucleotide (ODN) with a preference for the human Toll-like receptor 9 (TLR9). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides (CpG motifs). These unmethylated CpG motifs mimic microbial DNA and act as immunostimulants via TLR9. 

 More details

Mode of action

Stimulatory CpG ODNs are internalized and activate the endosomal receptor TLR9. Activation of TLR9 triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA and mitochondrial self-DNA [3, 4].

CpG Class A ODNs, such as ODN 2216, are characterized by a phosphodiester (PO) central CpG-containing palindromic motif and a phosphorothioate (PS)-modified 3’ poly-G string. 
InvivoGen's ODN 2243 contains GpC dinucleotides instead of CpGs and does not induce TLR9 activity [5-7].

In HEK-Blue™ hTLR9 reporter cells, ODN 2243 does not activate hTLR9 when compared to the immunostimulatory ODN 2216 (see figure).

Key features of ODN 2243

• Negative control of the human TLR9-activating ODN 2216
• Synthetic ODN with unmethylated GpC motifs
• Each lot is functionally tested

Get more information about CpG ODNs Classes.

Read our review on TLR9 agonists: double-edged sword for immune therapies.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Krieg A.M. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.
6. Vollmer J. et al., 2004. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur. J. Immunol. 34: 251–62.
7. Rothenfusser S. et al., 2004. CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro. Blood 103:2162-9.
8. Ayash-Rashkovsky M. et al., 2005. Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection. FASEB J. 19:1152-4.

Figures

Dose-dependent NF-κB response in HEK-Blue™ hTLR9 cells. HEK-Dual™ hTLR9 cells were incubated with increasing concentrations of ODN 2216 or the control ODN 2216 (ODN 2243). After 24h, the hTLR9-induced NF‑κB response was assessed by measuring the SEAP activity using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean + SEM).

Specifications

Specificity: Negative control of human TLR9-preferred ODN 2216 

Working concentration: 1-5 µM

Solubility:  5 mg/ml in water

ODN 2243 (ODN 2216 Control) sequence: 5’- ggGGGAGCATGCTGgggggg -3’ (20 mer)
Note: Bases shown in capital letters are phosphodiester, and those in lower case are phosphorothioate (nuclease resistant).

Quality control:

• The absence of TLR9 activity has been tested using HEK-Blue™ TLR9 cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

• 1 mg (154.50 nmol) lyophilized ODN 2243 (ODN 2216 Control)
• 1.5 ml sterile endotoxin-free water

ODN 2243 (ODN 2216 Control) is shipped at room temperature.

Upon receipt, store at -20°C.

Details

CpG ODNs

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 1018, have been extensively studied as adjuvants [1]. These CpG motifs are present at a 20-fold greater frequency in bacterial DNA compared to mammalian DNA [2]. CpG ODNs are recognized by the Toll-like receptor 9 (TLR9), which is expressed on human B cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th1-dominated immune responses [3]. Pre-clinical studies, conducted in rodents and non-human primates, as well as human clinical trials, have demonstrated that CpG ODNs can significantly improve vaccine-specific antibody responses [1]. Three types of stimulatory CpG ODNs have been identified, types A, B, and C, which differ in their immune-stimulatory activities [4-5]. 

Toll-like receptor 9

The Toll-like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [6-8]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [8,9]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [6,8].

TLR9 is mainly expressed in subsets of Dendritic Cells and B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [8]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [8]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [10].
 

References:

1. Steinhagen F. et al., 2011. TLR-based immune adjuvants. Vaccine 29(17):3341-55.
2. Hemmi H. et al., 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-5.
3. Coffman RL. et al., 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity 33(4):492-503.
4. Krug A. et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7): 2154-63.
5. Marshall JD. et al., 2005. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol. 24(2):63-72.
6. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
7. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
8. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
9. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
10. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.